The long term objective of this proposal is to gain a better understanding of how Mycobacterium tuberculosis establishes infection so that effective strategies can be developed to prevent it. A multidisciplinary approach will focus on the interaction of tubercle bacilli with macrophages at the molecular, genetic and cellular levels, with an emphasis on M. tuberculosis gene expression within macrophages. The specific aims are: 1) Identifying M. tuberculosis genes that are induced when bacteria are within macrophages by 2D gel electrophoresis coupled with mass spectrometry. 2) Identifying class-specific regulatory motifs among intracellular induced promoters using a combination of molecular and computational techniques. 3) Characterizing the roles in the M. tuberculosis-macrophage interaction of selected intracellular induced M. tuberculosis genes. Assays will include bacterial survival, replication and trafficking in macrophages. 4) Assessing the role of cAMP signaling in M. tuberculosis within macrophages by defining the distribution and expression among mycobacteria of genes encoding novel cyclase and cyclic NMP binding proteins; estimating the minimum number of cAMP-responsive proteins using 2D gels; and determining the effects of a novel adenylate cyclase gene knock-out on M. tuberculosis interaction with macrophages using tissue culture, microscopy, and 2D gel analyses. This work will contribute to our understanding of the factors needed for the establishment of tuberculosis infection and disease and will identify potential targets for tuberculosis vaccines, therapeutics, and diagnostic purposes.